Biochemistry 2004, 43, 6865-6876
6865
Serine Hydroxymethyltransferase: Role of Glu75 and Evidence that Serine Is Cleaved by a Retroaldol Mechanism† Doletha M. E. Szebenyi,‡ Faik N. Musayev,§ Martino L. di Salvo,| Martin K. Safo,§ and Verne Schirch*,| MacCHESS at Cornell High Energy Synchrotron Source, Cornell UniVersity, Ithaca, New York 14853, and Institute for Structural Biology and Drug DiscoVery and Department of Biochemistry, Virginia Commonwealth UniVersity, Richmond, Virginia 23219 ReceiVed January 29, 2004; ReVised Manuscript ReceiVed March 29, 2004
ABSTRACT: Serine hydroxymethyltransferase (SHMT) catalyzes the reversible interconversion of serine and glycine with tetrahydrofolate serving as the one-carbon carrier. SHMT also catalyzes the folateindependent retroaldol cleavage of allothreonine and 3-phenylserine and the irreversible conversion of 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate. Studies of wild-type and site mutants of SHMT have failed to clearly establish the mechanism of this enzyme. The cleavage of 3-hydroxy amino acids to glycine and an aldehyde occurs by a retroaldol mechanism. However, the folate-dependent cleavage of serine can be described by either the same retroaldol mechanism with formaldehyde as an enzyme-bound intermediate or by a nucleophilic displacement mechanism in which N5 of tetrahydrofolate displaces the C3 hydroxyl of serine, forming a covalent intermediate. Glu75 of SHMT is clearly involved in the reaction mechanism; it is within hydrogen bonding distance of the hydroxyl group of serine and the formyl group of 5-formyltetrahydrofolate in complexes of these species with SHMT. This residue was changed to Leu and Gln, and the structures, kinetics, and spectral properties of the site mutants were determined. Neither mutation significantly changed the structure of SHMT, the spectral properties of its complexes, or the kinetics of the retroaldol cleavage of allothreonine and 3-phenylserine. However, both mutations blocked the folate-dependent serine-to-glycine reaction and the conversion of methenyltetrahydrofolate to 5-formyltetrahydrofolate. These results clearly indicate that interaction of Glu75 with folate is required for folate-dependent reactions catalyzed by SHMT. Moreover, we can now propose a promising modification to the retroaldol mechanism for serine cleavage. As the first step, N5 of tetrahydrofolate makes a nucleophilic attack on C3 of serine, breaking the C2-C3 bond to form N5-hydroxymethylenetetrahydrofolate and an enzyme-bound glycine anion. The transient formation of formaldehyde as an intermediate is possible, but not required. This mechanism explains the greatly enhanced rate of serine cleavage in the presence of folate, and avoids some serious difficulties presented by the nucleophilic displacement mechanism involving breakage of the C3-OH bond.
Serine hydroxymethyltransferase (SHMT)1 catalyzes the reversible interconversion of serine and glycine with the hydroxymethyl group of serine (C3) being transferred to H4PteGlun to form CH2-H4PteGlun (1). This reaction serves as † This study was supported in part by U.S. Public Health Service Grant DK56648 to V.S. and by NIH NCRR Division Grant RR-01646 to MacCHESS. Some experimental work was carried out at CHESS, which is supported by NSF Grant DMR97-13424. * To whom correspondence should be addressed: Department of Biochemistry, 1101 E. Marshall St., Box 614, Virginia Commonwealth University, Richmond, VA 23298. E-mail:
[email protected]. Phone: (804) 828-9482. Fax: (804) 828-3093. ‡ Cornell University. § Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University. | Department of Biochemistry, Virginia Commonwealth University. 1 Abbreviations: SHMT, serine hydroxymethyltransferase; rcSHMT, rabbit cytosolic SHMT; scSHMT, sheep cytosolic SHMT; hcSHMT, human cytosolic SHMT; mcSHMT, mouse cytosolic SHMT; eSHMT, E. coli SHMT; bsSHMT, B. stearothermophilus SHMT; PLP, pyridoxal 5′-phosphate; H4PteGlu, tetrahydrofolate; CH2-H4PteGlu, 5,10-methylene-H4PteGlu; CH+-H4PteGlu, 5,10-methenyl-H4PteGlu; 5-CHO-H4PteGlu, 5-formyl-H4PteGlu; CHOH-H4PteGlu, N5-hydroxymethyleneH4PteGlu; SHMT‚Quin, intermediate between enzyme and glycine absorbing near 500 nm.
the major source of one-carbon groups required for the biosynthesis of purines, thymidylate, methionine, and many neurotransmitters. SHMT contains tightly bound PLP, and it is the interplay of H4PteGlun with PLP that is of great interest in terms of both the catalytic mechanism and nutritional studies that show a link between these two vitamins. After the discovery of SHMT activity, the enzyme was named serine aldolase, because it was conceived that the mechanism of the enzyme involved a retroaldol cleavage of serine to form formaldehyde and glycine (Figure 1a). In a second step, the bound formaldehyde was thought to condense with H4PteGlun to form CH2-H4PteGlun (Figure 1b). This proposed mechanism was strengthened when it was discovered that SHMT catalyzes, without the need for H4PteGlu, the rapid retroaldol cleavage of threonine and β-phenylserine to glycine with acetaldehyde and benzaldehyde, respectively (2, 3). SHMT has also been shown to catalyze the conversion of 5,10-CH+-H4PteGlun to 5-CHO-H4PteGlun (Figure 1c) (4); in this case, the 1-C group is at the oxidation level of formate
10.1021/bi049791y CCC: $27.50 © 2004 American Chemical Society Published on Web 05/11/2004
6866 Biochemistry, Vol. 43, No. 22, 2004
Szebenyi et al.
FIGURE 1: Proposed mechanisms for reactions catalyzed by SHMT and the requirement for general acid-base catalytic groups, labeled B1-B7. (a) Retroaldol mechanism for conversion of the serine internal aldimine to the glycine aldimine with formation of formaldehyde. E-Quin is the quinonoid intermediate and absorbs near 500 nm with a molar absorption coefficient of ∼40000 cm-1 M-1. B1 is the putative base required to accept the proton from the C3 hydroxyl group. (b) Condensation of formaldehyde (generated as shown in panel a) with H4PteGlu to form CH2-H4PteGlu. Only a portion of the H4PteGlu structure is shown. The carbinolamine and iminium cation are intermediates in the formation of CH2-H4PteGlu. (c) Proposed mechanism for conversion of CH+-H4PteGlu to 5-CHO-H4PteGlu with the formation of the intermediate CHOH-H4PteGlu. This reaction requires glycine as a ligand even though the glycine is not consumed. (d) Nucleophilic displacement mechanism for the serine to glycine reaction. As in panel a, the first structure is the external aldimine of serine. It is not known at what wavelength the putative covalent adduct labeled E-I would absorb, but from its structure it is most likely around 425 nm. The intermediate labeled E-Quin is the same structure shown in panel a. (e) A concerted mechanism that combines the reactions shown in panels a and b. Formaldehyde may be formed transiently in this mechanism if breaking of the bond between C2 and C3 of serine precedes formation of a bond between N5 of H4PteGlu and C3 of serine.
Serine Hydroxymethyltransferase instead of formaldehyde. Subsequent studies have provided evidence that the formation of 5-CHO-H4PteGlun plays an important regulatory role in 1-C metabolism (5-7). Three-dimensional structures have been determined for the cytosolic isozymes of human, rabbit, and mouse SHMT, as well as for SHMT from the prokaryotes Escherichia coli and Bacillus stearothermophilus (8-12). The active sites from these structural studies show conservation of key residues. Many of these residues have been changed by site-directed mutagenesis, and their role in binding and catalysis has been discussed. It has been suggested that the residues involved in the condensation of formaldehyde with H4PteGlun to form CH2-H4PteGlun (Figure 1b; the second step in folatedependent conversion of serine to glycine) are the same as in the hydrolysis of CH+-H4PteGlun to 5-CHO-H4PteGlun (Figure 1c) (13, 14). However, structural studies have been inconclusive regarding the role of various residues in reactions catalyzed by SHMT, and have not established whether formaldehyde is involved as an intermediate. Stereochemical considerations have raised doubts that formaldehyde is an intermediate, and the structure of a complex of serine with B. stearothermophilus SHMT supported a proposed mechanism in which N5 of H4PteGlun makes a nucleophilic displacement of the serine hydroxyl to form a covalent adduct between H4PteGlun and serine (Figure 1d) (12, 15). This intermediate collapses into products without the involvement of formaldehyde as an intermediate. Key differences between the retroaldol mechanism and this nucleophilic displacement mechanism include the geometry of reactive groups around the serine, the presence or absence of free formaldehyde, and the role of acid-base groups. The purpose of this study is to elucidate the role of Glu75 in rcSHMT by site-directed mutagenesis and structure determination. Glu75 could act as a proton donor or acceptor; it is situated at the active site, within hydrogen bonding distance of the C3 serine hydroxyl in a serine-SHMT complex and of the formyl oxygen of 5-CHO-H4PteGlu in a complex of this species with SHMT. Spectral and kinetic work on several mutants of Glu75 in scSHMT has determined that this residue plays an important role in the mechanism of the folate-dependent serine-to-glycine reaction (16), and structural studies on eSHMT suggest that Glu75 (Glu57 in eSHMT) is protonated (11). The work presented here confirms the importance of Glu75 in the folatedependent cleavage of serine (as a general acid-base catalyst) and establishes that it is not required for folateindependent allothreonine cleavage. In addition, it is clear that Glu75 is essential for the conversion of CH+-H4PteGlun to 5-CHO-H4PteGlun. The results are discussed in view of possible mechanisms for these reactions. EXPERIMENTAL PROCEDURES Materials. All amino acids, coenzymes, antibiotics, and buffers were obtained from Sigma (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA). (6S)-H4PteGlu and (6S)-5-CHOH4PteGlu were gifts from Eprova AG (Schaffhausen, Switzerland). Crystallization buffers were from Hampton Research (Laguna Niguel, CA). Site-Directed Mutagenesis. Mutants of rcSHMT were made using the QuickChange site-directed mutagenesis kit from Stratagene (La Jolla, CA) on rcSHMT cDNA in the
Biochemistry, Vol. 43, No. 22, 2004 6867 pET22b vector as described previously (17). Each mutation was confirmed by sequencing the cDNA insert in both directions. Each mutant protein was expressed in an E. coli glyA- strain as previously described (17). Purification was done by the same procedure published previously and resulted in high yields of >95% pure enzymes that exhibited the size and spectral characteristics of wild-type rcSHMT. Kinetic Studies. All spectra and steady state kinetic studies were performed in a cell with a path length of 1 cm with an Agilent 8354 spectrophotometer at 30 °C. The buffer was either 20 mM potassium phosphate or sodium N,N-bis(2hydroxyethyl)-2-aminoethane sulfonate (pH 7.3) unless otherwise noted. Catalytic activities for the cleavage of serine in the presence of H4PteGlu and allothreonine in the absence of H4PteGlu were performed as previously described (18). Dissociation constants2 for serine, glycine, and 5-CHOH4PteGlu were determined from double-reciprocal plots of the change in absorbance at 498 nm versus the change in the ligand concentration (19). The ligand concentrations were varied over a 10-fold range that bracketed the respective Kd values. For determination of the Kd for 5-CHO-H4PteGlu, the glycine concentration was held constant at 100 mM. The enzymatic rate of conversion of 5,10-CH+-H4PteGlu to 5-CHO-H4PteGlu was determined by adding 50 mM glycine to the reaction mixture and observing the increase in absorbance at 502 nm, where the SHMT‚Gly‚5-CHO-H4PteGlu ternary complex absorbs with a molar absorption coefficient of 40 000 M-1 cm-1 (2, 3). These reactions were performed at 20 °C because the increase in A502 was much greater upon formation of the ternary complex than at higher temperatures. The rate of conversion of L-serine to enzyme-bound glycine and formaldehyde was determined on an Applied Photophysics SX.18MV stopped-flow spectrophotometer. SHMT (9.0 mg/mL) was flowed against 200 mM L-serine at 30 °C, and the rate of formation of the SHMT‚Quin complex absorbing at 498 nm was observed. The kinetic traces were fit using software provided by Applied Photophysics. The rate of formaldehyde release was followed by trapping the formaldehyde with Schiff’s reagent and determining the absorbance at 550 nm as previously described (20). A standard curve of formaldehyde and excess Schiff’s reagent was used to convert absorbance into nanomoles of formaldehyde. Crystallization. Crystallization was performed at room temperature by the hanging drop vapor diffusion technique. Crystals of both mutants were obtained by mixing 3 µL of an enzyme solution [34-44 mg/mL in 20 mM potassium phosphate (pH 7.3) with 1 mM dithiothreitol] with an equal volume of reservoir solution. The reservoir solution for E75L rcSHMT consisted of 50 mM potassium phosphate (pH 6.6), 8.5-9.1% PEG 8000, and 100 mM KCl. For E75Q rcSHMT, the reservoir solution consisted of 15 mM potassium 2-(Nmorpholino)ethanesulfonate (pH 6.4), 8.5-10% PEG 4000, and 30 mM KCl. Serine complexes were formed by adding 0.25 µL of 250 mM L-serine directly to the drop, giving an ∼10 mM L-serine solution. Each mutant enzyme and the serine complexes formed yellow tetragonal bipyramidal crystals that reached a maximum size of ∼0.7 mm × ∼0.4 2 These are apparent Kd values as at least two steps are involved in forming the E‚Quin complex.
6868 Biochemistry, Vol. 43, No. 22, 2004 mm × 0.2 mm in 2-3 days. To obtain a ternary complex, crystals formed from the enzyme and serine were soaked for a few minutes in a solution containing 1 mM (6S)-H4PteGlu. Prior to data collection, crystals were washed with a solution of 8% PEG 4000, 100 mM KCl, and 50 mM potassium phosphate (pH 6.6). The wash was followed by a quick transfer to a series of solutions containing 15, 23, and 27% PEG 4000 and finally 27% PEG 4000 and 23% glycerol. The crystals were then rapidly frozen in liquid N2. Determination of the Structure. Data were collected from crystals of the E75Q and E75L mutants, with and without serine present; a partial data set was also obtained for E75L rcSHMT crystals soaked in serine and H4PteGlu (E75L-SerH4PteGlu set). All crystals were isomorphous with each other and with wild-type crystals; differences in unit cell dimensions were slight. The space group was P41212. Images were integrated with mosflm (21) and scaled with scala (22), using the DPS (23) interface. Initial structures were determined by rigid body refinement of either the wild-type structure or a previously refined mutant structure; the crystallographic R at this stage was between 0.23 and 0.30, depending on the degree of similarity between the starting model and the structure being determined. Manual adjustments in the region of the mutation were made, and refinement was carried out using CNS (24) (for grouped B-factor refinement and atomic position refinement using energy minimization) and O (25) (for manual adjustments, and addition of water molecules). Multiple cycles of refinement, and placement of water molecules, were carried out for the two higher-resolution data sets (E75L-Ser and E75Q). These structures were then used as starting points for the corresponding lower-resolution structures (E75L and E75L-Ser-H4PteGlu, and E75Q-Ser, respectively). In the case of E75L-Ser-H4PteGlu, the data were insufficient for complete refinement; a single cycle was carried out, followed by calculation of a difference map. Accurate determination of this structure will require further data collection. For E75L and E75Q-Ser, refinement was successfully carried out, although no attempt was made to modify the solvent structure in these lower-resolution cases. Final difference electron density maps for the four refined structures did not contain any significant features. The mammalian SHMT structure is a homotetramer, which may be described as a dimer of dimers. In the structures presented here, the asymmetric unit contains one tight dimer, made up of chains A and B; the second dimer is related to the first by a crystallographic symmetry operation. We use a residue numbering scheme based on the human SHMT sequence to facilitate comparison with other mammalian enzymes. With this scheme, each final structure includes residues 15-275 and 283-484 in chain A and residues 15277 and 282-484 in chain B. Missing residues, at the N-terminus and in the middle of a flexible loop, do not appear to play any role in molecular function. The dimer also includes two PLP molecules, each of which is primarily associated with one chain but also contacts residues from the second chain. The amino acid binding site involving the PLP primarily associated with chain A is designated site I, and the second site is site II. Approximately 100 water molecules are included, and one phosphate ion is present in most of the amino acid binding sites. E75Q has three water molecules in one of its amino acid binding sites and a phosphate ion in the other. For E75Q-
Szebenyi et al. Ser, an omit map calculated with omission of these solvent molecules clearly indicated the presence of glycine (or possibly disordered serine) in one of the two sites, and subsequent refinement included a Gly in this position. The amino acid binding sites of E75L-Ser contain primarily PO42-, and the E75L model included these ions from the beginning. The original determination of the wild-type rcSHMT structure (9) was in space group P41212, with one tight dimer per asymmetric unit. In a subsequent, higher-resolution, structure of an rcSHMT-H4PteGlu complex, the lower P41 symmetry was found, with a pair of dimers in the asymmetric unit (26). To verify the space group for these crystals, the data were reprocessed in space group P41. There was no significant improvement in R-factors, and the conformation of the two dimers remained the same. We conclude that, to the resolution of the current data, P41212 symmetry is preserved for the E75L and E75Q mutants. Statistics from data collection and processing, and structure determination, are summarized in Table 1. Coordinates have been deposited in the Protein Data Bank (27), as entries 1RV3 (E75L-Ser), 1RV4 (E75L), 1RVU (E75Q), and 1RVY (E75Q-Ser). RESULTS Spectral Properties of rcSHMT Mutants E75Q and E75L. The mutant enzymes were expressed and purified by the same procedure used for recombinant wild-type rcSHMT. The enzymes are yellow, exhibiting a single absorption maximum above 300 nm at 428 nm with a 278 nm:428 nm ratio of 6.2. The mutant enzymes were stable at -20 °C for several months. Glycine, added to saturating conditions, resulted in three observable absorption maxima at 498, 425, and 343 nm. These absorption maxima reflect three intermediates on the reaction pathway. The 343 nm peak is the gem-diamine, which occurs as the first covalent complex of the enzyme (28). This is followed by formation of the external aldimine complex, absorbing at 425 nm, which is shown as the first and last structures in Figure 1a for serine and glycine, respectively. The 498 nm-absorbing species is the quinonoid complex, in which a pair of electrons on C2 of glycine are in resonance with the π-electron system of the bound pyridoxal phosphate (28). This structure is labeled E‚Quin in Figure 1a. The spectra of both the E75Q and E75L glycine complexes are very similar to the spectrum of the wild-type rcSHMT‚Gly complex. The major difference between the mutant and wild-type enzymes is that the absorption peak near 500 nm is severalfold higher. As with the wild-type rcSHMT‚Gly complex, lower temperatures and higher pH values shift the equilibrium from the aldimine complex to the gem-diamine complex. By titrating the enzyme with glycine and determining the increase in absorption at 498 nm, we were able to determine a Kd value for glycine for each mutant rcSHMT (Table 2). The affinity for glycine was nearly normal for E75Q, but was almost 8-fold lower for E75L. The addition of either H4PteGlu or 5-CHO-H4PteGlu to the mutant rcSHMT‚Gly complexes resulted in a severalfold increase in the absorption of the 500 nm peak at 20 °C. This is also observed with wild-type rcSHMT. This spectral effect of 5-CHO-H4PteGlu permitted a determination of the Kd value for 5-CHO-H4PteGlu for both E75Q and E75L
Serine Hydroxymethyltransferase
Biochemistry, Vol. 43, No. 22, 2004 6869
Table 1: Summary of Data Collection, Data Reduction, and Structure Refinementa E75L unit cell dimensions a, c (Å) resolution (Å) Rsym I/σ(I) completeness (%) redundancy no. of observations no. of unique reflections R/Rfree average B total no. of reflections no. of reflections in the test set total no. of non-H atoms no. of water molecules rmsd from ideal geometry bond lengths (Å) bond angles (deg)
E75L-Ser
E75L-Ser-H4PteGlu
Data Collection and Initial Processing 115.0, 158.4 114.9, 158.3 114.7, 158.0 36-2.95 49-2.40 49-2.95 0.116 (0.553) 0.100 (0.566) 0.166 (0.669) 6.1 (1.4) 6.0 (1.3) 3.4 (1.0) 99.9 99.8 73.4 9.0 13.0 5.3 207374 547673 87974 23067 42006 16463 0.201/0.245 50 22868 1113 7310 86 0.007 1.35
Structure Determination and Refinement 0.224/0.255 0.226/0.310 49 66 41555 16382 2087 795 7310 7344 86 86 0.007 1.34
0.008 1.33
E75Q
E75Q-Ser
114.6, 156.8 58-2.50 0.111 (0.544) 6.4 (1.4) 99.6 5.3 195522 36699
114.7, 156.4 27-2.90 0.188 (0.626) 3.8 (1.1) 93.8 5.8 130399 22342
0.221/0.253 37 36655 1843 7326 105
0.220/0.283 34 22026 1242 7328 102
0.007 1.32
0.007 1.34
a Data were collected at the Cornell High Energy Synchrotron Source (CHESS), station A-1, for E75L, E75L-Ser, and E75L-Ser-H PteGlu 4 crystals, and on a rotating anode generator for E75Q and E75Q-Ser crystals. A single crystal was used for each collection, except for E75L-SerH4PteGlu, for which two crystals were used. The detector was an ADSC Quantum-210 instrument at CHESS and a Rigaku R-axis instrument in the home lab. Values of Rsym and I/σ(I) are given as the overall values (values for the highest-resolution shell in parentheses).
Table 2: Kinetic Constants for rcSHMT Mutant Enzymes (30 °C) constant Km (Ser) (mM) Kd (Gly) (mM) Kd (5-CHO-H4PteGlu) (µM) kcat (Ser) (min-1) Km (alloThr) (mM) kcat (alloThr) (min-1) kE‚Quin (min-1) kHCHO (min-1)
wild-type rcSHMT 0.8 6 10 600 1.5 130 not determined 0.0028d
E75L
E75Q
45 26 not detectable 22 144 (110%)b 64 and 23c 0.34d
4.0 7.7 10 1.2 15 560 (430%)b 460 and 11 0.13
27a
a This is a K value. b Percentage of the wild-type value in parentheses. c Biphasic rate constants for formation of E‚Quin from serine absorbing d at 498 nm. d Estimate of the rate of formation of HCHO from serine as determined from the initial rate.
rcSHMT (Table 2). Compared to wild-type rcSHMT, E75L rcSHMT has a 2.5-fold lower affinity for 5-CHO-H4PteGlu while E75Q rcSHMT has the same affinity. Saturation of wild-type rcSHMT with L-serine also results in the three absorption maxima at 343, 425, and 498 nm, but the intensity of the 343 and 498 nm peaks is greatly diminished compared to those observed with glycine (28). However, with both E75Q and E75L rcSHMT, saturation with serine results in significant absorption maxima at 343 and 498 nm (the absorption maximum with E75L rcSHMT is actually at 502 nm) (Figure 2). Again, at lower temperatures or higher pH, the equilibrium is shifted toward the gem-diamine intermediate absorbing at 343 nm. Titration of E75Q and E75L rcSHMT with serine permitted determination of their respective Kd values via a plot of the reciprocal increase in A498 against the reciprocal serine concentration (Table 2). E75Q and E75L rcSHMTs had 5- and 30-fold reduced affinity for serine, respectively, compared to the wild-type enzyme. Kinetic Constants for E75Q and E75L rcSHMT with Serine and Allothreonine. Attempts were made to determine Km and kcat values for the conversion of serine and H4PteGlu to glycine and 5,10-CH2-H4PteGlu by the normal assay procedure. With E75L rcSHMT, there was no detectable activity, but with a high concentration of E75Q, activity was observed and a kcat value of 1.2 min-1 was determined, assuming that
FIGURE 2: Spectra of E75L rcSHMT as a function of serine concentration and temperature: (s) 1.2 mg/mL E75L rcSHMT at pH 7.6, (‚‚‚) after the addition of L-serine to a final concentration of 60 mM at 7 °C, and (- - -) same solution but at 37 °C.
both serine and H4PteGlu were at saturating concentrations (serine at 50 mM and H4PteGlu at 0.2 mM) (Table 2). This represents a 500-fold decrease in activity compared to that of the wild-type enzyme. In contrast, both mutant enzymes exhibited greater catalytic activity with allothreonine in the absence of H4PteGlu than the wild-type enzyme (Table 2). The Km values for allothreonine are increased by a factor of
6870 Biochemistry, Vol. 43, No. 22, 2004 ∼10, while the kcat value for E75Q rcSHMT is more than 4-fold higher than that of the wild-type enzyme. If allothreonine is processed by the retroaldol cleavage mechanism shown in Figure 1a, and serine (in the presence of H4PteGlu) is processed by the same mechanism followed by the condensation of formaldehyde with H4PteGlu (Figure 1b), then these results strongly suggest that Glu75 is associated with the condensation step and not with the retroaldol cleavage step. If the mechanisms are not the same, Glu75 is clearly implicated in the one which operates in the presence of folate. We had previously investigated the ability of rcSHMT to cleave serine to glycine and formaldehyde in the absence of H4PteGlu (29). We showed that this did occur very slowly, with a kcat of 0.0028 min-1. Evidence suggested that the ratedetermining step was the release of formaldehyde and not serine cleavage (30). Both E75Q and E75L rcSHMT saturated with serine show a significant absorption peak near 500 nm that is not seen with wild-type rcSHMT at pH
